Summary/Abstract Precise separation of replicated genetic material during cell division is required for the generation, development and survival of all organisms. Segregation of this replicated genetic material, or chromosomes, relies on the correct timing and location of attachment to a conserved megadalton-sized protein network called the kinetochore. Once attached to the kinetochore, duplicated chromosomes are pulled apart to be distributed evenly to resulting daughter cells after cell division. Errors in this process can result in the rapid accumulation of mis- segregated chromosomes resulting in a cellular condition called aneuploidy, a hallmark of cancerous cells. To ensure productive kinetochore attachments that yield proper segregation of chromosomes, the initiation and maintenance of kinetochore assembly is tightly regulated in cells. Despite high conservation of the kinetochore protein scaffold among eukaryotes, the fundamental mechanics of the initiation and regulation of this process are not well understood. This proposal aims to use an interdisciplinary approach that integrates yeast genetics, molecular biology, protein biochemistry, and single-molecule imaging to address several key outstanding questions: to determine the regulation and dynamics of inner kinetochore assembly, and to elucidate key phosphorylation sites that regulate kinetochore initiation. Using a recently developed technique of real-time monitoring of kinetochore assembly in Saccharomyces cerevisiae via colocalization spectroscopy, this project will first map the precise dynamics, and regulation of kinetochore assembly initiation. This will be accomplished by monitoring the first steps of kinetochore formation, deposition of the histone variant protein Cse4 onto centromeric DNA in real-time. In tandem, this project will rely on a novel technique of de novo assembly of native kinetochores on centromeric DNA to determine the role of phosphorylation and associated regulatory mechanisms in Cse4 deposition and kinetochore assembly initiation. Together, these studies will rigorously determine how kinetochore assembly is initiated in molecular detail. Importantly, these details will provide a framework to better understand potential mechanisms of cancer initiation and progression that are critical for future development of therapies to treat this devastating disease. Through the mentorship and collaboration facilitated by this fellowship, I will gain valuable expertise in the field of kinetochore biology as well as an understanding of how to address key outstanding questions in the field. This training, coupled to my experience during my graduate study with recombinant proteins, genetic code expansion, and single molecule microscopy, will provide a research foundation such that I will be prepared to perform independent research focused on elucidating the mechanisms that regulate mitotic spindle function to drive chromosome separation during cell division. Additionally, the Fred Hutchinson Cancer Research Center is an ideal environment for the proposed studies due to access to leading technologies and resources as well as a highly interactive scientific environment with surrounding experts in biochemistry and biophysics.